The Endocrine Society guideline on Androgen Deficiency in Men emphasized that accurate measurement of testosterone (T) levels is central to the diagnosis of androgen deficiency. However, the accuracy of direct radioimmunoassays for the measurement of total T levels has been questioned. Furthermore, reference limits for total and free T levels generated in a population-based sample of community-dwelling men are not available. In the absence of standardized reference limits, the partitioning of total and free T levels into normal or low values is fraught with substantial risk of misclassification. The objective of this collaboration among investigators from BUMC, Framingham Heart Study (FHS), CDC, and Mayo Clinic is to generate reference limits for total and free T levels in a sample of healthy young men in the community- based FHS third generation (Gen 3) cohort. Total T levels will be measured by liquid chromatography tandem mass spectrometry (LC-MS/MS), using a CDC calibrator. We will calculate free T from total T and SHBG, by using law of mass action equation, in accordance with Endocrine Society's position statement. We will generate reference limits for total and free T levels using nonparametric methods stratifying by age and apply the reference limits generated in Gen 3 sample to the distribution of values in a broad sample of men in both Gen 3 and Offspring (Gen 2) cohorts. We will evaluate the cross-sectional clinical correlates of total and free T levels in the broad sample using multivariable regression. adjusting for age, BMI, co-morbid conditions, smoking, and physical activity. Longitudinally, we will relate total and free T levels at baseline (examination 7) to the incidence of adverse outcomes (primary: mortality, diabetes mellitus, cardiovascular disease events (CVD);secondary: progression of functional limitations and disability) over a follow-up of 10 years. We will develop recommendations for partitioning of men into those with low or normal levels, based on considerations of statistical distribution of total and free T values and the association of outcomes with varying degree of deviations from the reference limits. The proposed study will provide a framework for the interpretation of serum T levels and enhance the comprehensibility of circulating T values to practicing clinicians. These steps will facilitate the development of rational criteria for classifying men into androgen-deficient and androgen-replete categories.